Various methods for analysing nucleic acid sequences are known in the art. Often, such techniques involve amplification—such as by PCR—of one or more parts of the nucleic acid(s) of a mixture of restriction fragments generated from the nucleic acid(s). The amplified mixture thus obtained is then analysed, e.g. by detection of one or more of the amplified fragments. For example, the amplified fragments may be separated on the basis of differences in length or molecular weight, such as by gel-electrophoresis, after which the amplified fragments are visualised, e.g. by autoradiography of the labelled amplified fragments or blotting followed by hybridisation. The resulting pattern of bands is referred to as a “(DNA) fingerprint”.
Usually in DNA fingerprinting, fingerprints of closely related species, subspecies, varieties, cultivars, races or individuals are compared. Such related fingerprints can be identical or very similar, i.e. contain a large number of corresponding—and therefore less informative—bands. Differences between two related DNA-fingerprints are referred to as “DNA-polymorphisms”. These are amplified fragments—i.e. bands—, which are unique in or for a fingerprint and/or for a subset of fingerprints. The presence or absence of such polymorphic fragments in a fingerprint—or the pattern thereof—can be used as a genetic marker, for instance to identify a specific species, subspecies, variety, cultivar, race or individual; to establish the presence or absence of a specific inheritable trait and/or of a specific gene; and/or to determine the state of a disease. For a further discussion of DNA-fingerprinting, DNA-polymorphisms, genotyping, PCR and similar amplification techniques, as well as the techniques and materials used therein, reference is inter alia made to the prior art mentioned hereinbelow, as well as to the standard handbooks.
One DNA-fingerprinting technique—which is advantageous in that it requires no prior knowledge of the sequence to be analysed—is selective restriction fragment amplification or AFLP®. In general, AFLP® comprises the steps of:    (a) digesting a nucleic acid, in particular a DNA, with one or more specific restriction endonucleases, to fragment the DNA into a corresponding series of restriction fragments;    (b) ligating the restriction fragments thus obtained with a double-stranded synthetic oligonucleotide adapter, one end of which is compatible with one or both of the ends of the restriction fragments, to thereby produce tagged restriction fragments of the starting DNA;    (c) contacting the tagged restriction fragments under hybridising conditions with a oligonucleotide primer;    (d) amplifying the tagged restriction fragment hybridised with the primers by PCR or a similar technique so as to cause further elongation of the hybridised primers along the restriction fragments of the starting DNA to which the primers hybridised; and    (e) detecting, identifying or recovering the amplified or elongated DNA fragment thus obtained.
The AFLP-fingerprint thus obtained provides information on sequence variation m (subsets of) the restriction enzyme sites used for preparation of the AFLP template and the nucleotide(s) immediately adjacent to these restriction enzyme sites in the starting DNA. By comparing AFLP-fingerprints from related individuals, again polymorphic fragments (also referred to as “AFLP-markers”) can be detected/identified, e.g. for the purposes mentioned hereinabove.
For a further description of AFLP® its advantages, its embodiments, as well as the techniques, enzymes, adapters, primers and further compounds and tools used therein, reference is made to EP-A-0 534 858 and co-pending European applications 98.202.5496 and 98.202.4515, all by applicant.
However, although AFLP® is a very efficient technique for identifying and analysing polymorphisms in random subsets of nucleic acid sequences, it cannot be used to analyse nucleic acid sequences for polymorphisms/markers associated with particular sequences such as highly polymorphic microsatellites.
It is known that the genome of many—if not all—eukaryotic organisms contains a large number of repeating nucleotide sequences, which are variously referred to as “simple sequence repeats” or “SSRs”; “simple sequence length polymorphisms” or “SSLPs”; “dinucleotide, trinucleotide, tetranucleotide or pentanucleotide repeats”;“(short) tandem repeats” or “STRs”; and/or “microsatellites” (the term mainly used in the present description). It is also known that such microsatellites may provide codominant genetic markers with a high degree of allelic polymorphism. Consequently, microsatellites generally have a higher Polymorphism Information Content (PIC) than bi-allelic markers, such as most AFLP markers. Accordingly, various techniques have been developed in the art to analyse nucleic acid sequences for the presence or absence of such microsatellite-associated polymorphisms that may be used as genetic markers.
Usually, these known techniques involve the amplification of the nucleic acid or a mixture of restriction fragments generated from the nucleic acid with a combination of (at least) two primers, which either both flank the repeat motif of a microsatellite present in the starting nucleic acid, or of which at least one is (intended to be) complementary to a microsatellite sequence present in the starting nucleic acid (also referred to as the “microsatellite-directed primer”). The amplified mixture thus obtained is then analysed, for instance based upon the differences in length of the amplified fragments obtained. Usually, this is carried out using conventional gelelectrophoresis/autoradiography to provide a fingerprint, which can then be analysed for the presence or absence of the specific polymorphic fragments. Generally, these known techniques differ in the primers combinations used to amplify the nucleic acid fragments, as will be further discussed hereinbelow. For a description of some of these known techniques, reference is made to WO 96/22388 by applicant; EP 0 804 618; Wu et al., Nucl. Acids Research, 1994, Vol.22., No.15, 3257–3258; Matsumoto et al, Mammalian Genome 9, 531–535 (1998); as well as to some of the further prior art mentioned therein. In the state of the art, a method for analysing nucleic acid sequences for microsatellite-associated polymorphisms or markers therefor, involving the use of a combination of both a microsatellite-based RAMP-primer and an AFLP-primer has not yet been disclosed or suggested.
For instance, Wu et al. describe the use of a RAMP-primer in combination with an RAPD-primer. Such an RAPD primer differs from an AFLP-primer in that it is not (intended to be) complementary to an adapter. Thus, in the method of Wu et al, the target DNA will usually also not contain an adapter.
EP 0 804 618 describes a technique referred to as “SAMPL”. This technique involves the use of two primers, one of which may inter alia be an AFLP-primer, and the other of which—the microsatellite-directed primer also referred to as the “SAMPL”-primer—is (intended to be) complementary to a so-called “compound repeat SSR”, which is an SSR comprising at least two different parts with each part comprising a different repeated sequence (e.g. 5′-CTCTCTCTGAGAGAGA-3′). However, even though the latter primer is intended to be complementary to a microsatellite (albeit a specific type of microsatellite) and even though the primer may also be considered to comprise a 3′-part and a 5′-part, the microsatellite-directed primer used in EP 0 804 618 differs from the RAMP-primer used in the invention.
Thus, it is an object of the invention to provide improved or alternative methods for identification and analysis of microsatellite-based polymorphisms and markers based thereon. Particularly it is an object of the invention to provide such methods involving the use of a combination of both a microsatellite-based RAMP-primer and an AFLP-primer.